Compositions and methods for dental mineralization

ABSTRACT

The present invention relates to compositions for mineralizing a dental surface, in particular tooth enamel. Methods of mineralizing hypomineralized lesions (including subsurface lesions) in the tooth enamel caused by dental caries, dental corrosion and fluorosis are also provided. In particular, the invention relates to a method of mineralizing a dental surface or subsurface comprising contacting the dental surface or subsurface with a compound that is capable of increasing or maintaining the pH of a solution and a mineralizing agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.16/852,983 filed Apr. 20, 2020, which is a continuation of U.S.application Ser. No. 14/906,395, filed Jan. 20, 2016, which is the U.S.National Stage of International Application No. PCT/AU2014/050144, filedJul. 23, 2014, and claims priority to Australian Patent Application No.2013902815, filed Jul. 23, 2013.

FIELD OF THE INVENTION

The present invention relates to compositions for mineralizing a dentalsurface, in particular tooth enamel. Methods of mineralizinghypomineralized lesions (including subsurface lesions) in the toothenamel caused by various conditions such as dental caries, dentalcorrosion and fluorosis are also provided.

BACKGROUND OF THE INVENTION

Common causes of hypomineralized lesions are caries and fluorosis.

Dental caries result from the demineralization of hard tissue of theteeth usually because of fermentation of dietary sugar by dental plaqueodontopathogenic bacteria. Dental caries is still a major public healthproblem. Further, restored tooth surfaces can be susceptible to furtherdental caries around the margins of the restoration. Even though theprevalence of dental caries has decreased through the use of fluoride inmost developed countries, the disease remains a major public healthproblem. Dental erosion or corrosion is the loss of tooth mineral bydietary or regurgitated acids. Dental hypersensitivity is due to exposeddentinal tubules through loss of the protective mineralized layer,cementum. Dental calculus is the unwanted accretion of calcium phosphateminerals on the tooth surface. All these conditions, dental caries,dental erosion, dental hypersensitivity and dental calculus aretherefore imbalances in the level of calcium phosphates.

Enamel fluorosis (mottling) has been recognized for nearly a century,however, the aetiological role of fluoride was not identified until1942. The characteristic appearance of fluorosis may be differentiatedfrom other enamel disturbances. The clinical features of fluoroticlesions of enamel (FLE) represent a continuum ranging from fine opaquelines following the perikymata, to chalky, white enamel. The presence ofa comparatively highly mineralized enamel outer surface and ahypomineralized subsurface in the fluorotic lesion simulates theincipient enamel “white spot” carious lesion. With increasing severity,both the depth of enamel involved in the lesion and the degree ofhypomineralization increases. The development of fluorosis is highlydependent on the dose, duration and timing of fluoride exposure and isbelieved to be related to elevated serum fluoride concentrations. Chalky“white spot” lesions may also form on developing teeth in children suchas after treatment with antibiotics or fever. Such lesions indicateareas of hypomineralization (i.e. too little mineralization) of thetooth enamel.

Depending on lesion severity, fluorosis has been managed clinically byrestorative replacement or micro-abrasion of the outer enamel. Thesetreatments are unsatisfactory because they involve restorations orremoval of tooth tissue. What is desired is a treatment that willmineralize the hypomineralized enamel to produce a natural appearanceand structure.

Specific complexes of casein phosphopeptides and amorphous calciumphosphate (“CPP-ACP”, available commercially as Recaldent®) have beenshown to remineralize enamel subsurface lesions in vitro and in situ(Reynolds, 1998; Shen et al., 2001; Reynolds et al., 2003).

WO 98/40406 in the name of The University of Melbourne (the contents ofwhich are herein incorporated fully by reference) describes caseinphosphopeptide-amorphous calcium phosphate complexes (CPP-ACP) andCPP-stabilized amorphous calcium fluoride phosphate complexes (CPP-ACFP)which have been produced at alkaline pH. Such complexes have been shownto prevent enamel demineralization and promote remineralization ofenamel subsurface lesions in animal and human in situ caries models(Reynolds, 1998). Improved casein phosphopeptide-amorphous calciumphosphate complexes (CPP-ACP) and CPP-stabilized amorphous calciumfluoride phosphate complexes (CPP-ACFP) have also been described inWO2006/056013 and WO2006/135982.

The CPP which are active in forming the complexes do so whether or notthey are part of a full-length casein protein. Examples of active (CPP)that can be isolated after tryptic digestion of full length casein havebeen specified in U.S. Pat. No. 5,015,628 and include peptides Bosα_(s1)-casein X-5P (f59-79), Bos β-casein X 4P (f1-25), Bosα_(s2)-casein X-4P (f48-70) and Bos α_(s2)-casein X-4P (f1-21).

There is a need to provide improved or alternative treatments forhypomineralized lesions.

Reference to any prior art in the specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in Australia or any otherjurisdiction or that this prior art could reasonably be expected to beascertained, understood and regarded as relevant by a person skilled inthe art.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of mineralizing adental surface or subsurface comprising contacting the dental surface orsubsurface with a compound that is capable of increasing or maintainingthe pH of a solution and a mineralizing agent. Preferably, themineralizing agent is stabilized amorphous calcium phosphate (ACP)and/or amorphous calcium fluoride phosphate (ACFP). Typically, thecompound is capable of maintaining the pH of a solution between 7 to 9,preferably about 7.5. Preferably, the compound is provided in an amounteffective to raise the pH of intra-lesion fluid of a dental lesion from6 to 7.5. The compound is capable of increasing the pH of a solutionthat has an acidic pH (i.e. less than pH 7). The dental surface ispreferably dental enamel. In one embodiment the dental surface is alesion in the enamel, such as a lesion caused by caries, dental erosionor fluorosis.

A compound which is capable of increasing or maintaining the pH of asolution includes a compound which can accept hydrogen cations (protons)or, more generally, donate a pair of valence electrons. Preferably, thecompound is a base. The compound may not necessarily normally beregarded as a base, for example a polypeptide with numerous acidic andbasic residues but nonetheless has the ability to maintain or increasethe pH of a solution between 7 to 9, preferably 7.5. The compound iscapable of increasing the pH of a solution that has an acidic pH (i.e.less than pH 7). For example, the compound is capable of increasing thepH of the acidic intra-lesion fluid of a subsurface lesion requiringremineralisation. Preferably, the compound is provided in an amounteffective to raise the pH of intra-lesion fluid of a dental lesion from6 to 7.5. In one embodiment, a compound which is capable of increasingor maintaining the pH of a solution is an alkali which has the capacityto release hydroxide ions.

A compound which is capable of increasing or maintaining the pH of asolution also includes a compound that can maintain as a buffer the pHof a neutral or basic solution (i.e. pH greater than or equal to 7) whenthe neutral or basic solution is exposed to an acid. Typically, thecompound is capable of maintaining the pH of a solution between 7 to 9,preferably about 7.5.

Any pharmaceutically acceptable compounds described as a base aresuitable for use in the invention. Typically, the base is suitable fororal use. Preferably, the compound acts as a base, i.e. only releaseshydroxide ions or donates electrons, in the presence of an acid. Thebase may be a free-base form, or in a pharmaceutically acceptable saltform. Non-limiting examples of bases suitable for use in the inventioninclude hydroxides, chlorides, borates, phosphates including hydrogenphosphates and dihydrogen phosphates, citrates, carbonates,bicarbonates, hypochlorites (such as sodium hypochlorite), amines andany salt forms thereof including an alkali metal salt forms. Morespecifically, non-limiting examples of suitable pharmaceuticallyacceptable bases include ammonium hydroxide, sodium hydroxide, potassiumhydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide,ferrous hydroxide, zinc hydroxide, sodium hypochlorite, copperhydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine,ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine,arginine, histidine or urea. A hypofluorite capable of acting as a baseas described herein is also useful in the invention as the agent forincreasing or maintaining pH. A suitable hypofluorite would react insitu, being a surface or subsurface lesion, to produce fluoride ions andhydroxide (or another base) ions. As one skilled in the art willappreciate a potentially favourable outcome of the production offluoride ions is that fluoride ions can substitute for hydroxide in thecrystal structure of apatite forming fluorapatite.

Preferably the ACP and/or ACFP is phosphopeptide (PP)-stabilized.Preferably, the phosphopeptide (as defined below) is a caseinphosphopeptide. Preferably, the ACP or ACFP is in the form of a caseinphosphopeptide stabilized ACP or ACFP complex.

In a preferred embodiment, the phosphopeptide stabilized amorphouscalcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP)complex has tightly bound and loosely bound calcium, wherein the tightlybound calcium in the complex is less than the tightly bound calcium inan ACP or ACFP complex formed at a pH of 7.0. Optionally, the ACP orACFP is predominantly in a basic form.

In a preferred embodiment, the calcium ion content of the stabilized ACPor ACFP complex is in the range of about 30 to 100 moles of calcium permole of PP. More preferably, the calcium ion content is in the range ofabout 30 to about 50 moles of calcium per mole of PP.

In any aspect or embodiments as described herein, the stabilized ACPand/or ACFP may be in a formulation with additional calcium phosphate.Typically, the formulation includes a PP stabilized ACP and/or ACFPcomplex together with at least an equal amount by weight of calciumphosphate.

In a preferred embodiment the ACP and/or ACFP is in the form of a caseinphosphopeptide stabilized ACP and/or ACFP complex. Preferably, the phaseof the ACP is predominantly a basic phase, wherein the ACP comprisespredominantly the species Ca²⁺, PO₄ ³⁻ and OH⁻. The basic phase of ACPmay have the general formula [Ca₃(PO₄)₂]_(x)[Ca₂(PO₄)(OH)] where x≥1.Preferably x=1-5. More preferably, x=1. Preferably the two components ofthe formula are present in equal proportions. Accordingly, in oneembodiment, the basic phase of ACP has the formulaCa₃(PO₄)₂Ca₂(PO₄)(OH).

Preferably, the phase of the ACFP is predominantly a basic phase,wherein the ACFP comprises predominantly the species Ca²⁺, PO₄ ³⁻ and F.The basic phase of ACFP may have the general formula[Ca₃(PO₄)₂]_(x)[Ca₂(PO₄)F]_(y), where x≥1 when y=1 or where y≥1 whenx=1. Preferably, y=1 and x=1-3. More preferably, y=1 and x=1. Preferablythe two components of the formula are present in equal proportions.Accordingly, in one embodiment, the basic phase of ACFP has the formulaCa₃(PO₄)₂Ca₂(PO₄)F.

In one embodiment, the ACP complex consists essentially ofphosphopeptides, calcium, phosphate and hydroxide ions and water.

In one embodiment, the ACFP complex consists essentially ofphosphopeptides, calcium, phosphate, fluoride and hydroxide ions andwater.

In a further aspect of the present invention there is provided a methodof mineralizing a dental surface comprising providing a compound whichis capable of increasing or maintaining the pH of a solution and asource of ACP or ACFP. In a preferred embodiment the dental surface isenamel. Preferably the compound which is capable of increasing ormaintaining the pH of a solution is a base. The base may be any basedescribed herein, including, but not limited to, sodium bicarbonate,sodium hypochlorite, a hypofluorite or urea.

In a further aspect of the present invention there is provided a methodfor treating fluorosis comprising contacting a fluorotic lesion in toothenamel with a compound which is capable of increasing or maintaining thepH of a solution and stabilized ACP and/or ACFP. Preferably the compoundwhich is capable of increasing or maintain the pH of a solution is abase. The base may be any base described herein, including, but notlimited to, sodium bicarbonate, sodium hypochlorite, a hypofluorite orurea.

In a further aspect of the present invention there is provided a methodfor treating dental caries comprising contacting a caries lesion intooth enamel with a compound which is capable of increasing ormaintaining the pH of a solution and stabilized ACP and/or ACFP.Preferably the compound which is capable of increasing or maintain thepH of a solution is a base. The base may be any base described herein,including, but not limited to, sodium bicarbonate, sodium hypochlorite,a hypofluorite or urea.

In a further aspect of the present invention there is provided a methodfor treating dental erosion comprising contacting a lesion in toothenamel caused by erosion with a compound which is capable of increasingor maintaining the pH of a solution and stabilized ACP and/or ACFP.Preferably the compound which is capable of increasing or maintain thepH of a solution is a base. The base may be any base described herein,including, but not limited to, sodium bicarbonate, sodium hypochlorite,a hypofluorite or urea.

In a further aspect of the present invention there is provided a methodfor reducing white spot lesions on the tooth enamel comprisingcontacting a white spot lesion with a compound which is capable ofincreasing or maintaining the pH of a solution and stabilized ACP and/orACFP. Preferably the compound which is capable of increasing or maintainthe pH of a solution is a base. The base may be any base describedherein, including, but not limited to, sodium bicarbonate, sodiumhypochlorite, a hypofluorite or urea.

In a further aspect of the present invention there is provided a methodfor remineralizing a lesion in tooth enamel comprising contacting thelesion with a compound which is capable of increasing or maintaining thepH of a solution and stabilized ACP and/or ACFP. Preferably the compoundwhich is capable of increasing or maintain the pH of a solution is abase. The base may be any base described herein, including, but notlimited to, sodium bicarbonate, sodium hypochlorite, a hypofluorite orurea.

In a further aspect of the invention there is provided a method forremineralizing a lesion in tooth enamel comprising contacting the lesionwith stabilized ACP and/or ACFP followed by administering a compositioncontaining sodium bicarbonate or urea. Preferably, the composition is amouthrinse or mouthwash containing sodium bicarbonate or urea.

In one embodiment, the compound which is capable of increasing ormaintaining the pH of a solution is not sodium hypochlorite (NaOCl) anda composition of the invention is sodium hypochlorite (NaOCl) free.

In any aspect or embodiment of the invention described herein, thecompound which is capable of increasing or maintaining the pH of asolution is administered concurrently with, as a pre-treatment to, or asa post-treatment to a source of stabilized ACP or ACFP. Preferably thecompound which is capable of increasing or maintain the pH of a solutionis a base. The base may be any base described herein, including, but notlimited to, sodium bicarbonate, sodium hypochlorite, a hypofluorite orurea.

In any aspect or embodiment of the invention described herein, thecompound which is capable of increasing or maintaining the pH of asolution is administered concurrently with or as a post-treatment to asource of stabilized ACP or ACFP. Preferably the compound which iscapable of increasing or maintain the pH of a solution is a base. Thebase may be any base described herein, including, but not limited to,sodium bicarbonate, sodium hypochlorite, a hypofluorite or urea.

In any aspect or embodiment of the invention described herein, thecompound which is capable of increasing or maintaining the pH of asolution and stabilized ACP or ACFP is applied to the mouth, tooth orlesion by the subject in need of treatment or by a dental health careprofessional.

In any aspect or embodiment of the invention described herein, the ACPand/or ACFP is phosphopeptide (PP)-stabilized. Preferably, thephosphopeptide (as defined below) is a casein phosphopeptide.Preferably, the ACP or ACFP is in the form of a casein phosphopeptidestabilized ACP or ACFP complex.

The compound which is capable of increasing or maintaining the pH of asolution may be contacted with the dental surface for a period of about1 to 60 minutes, or for about 1 to 30 minutes. In one embodiment, thecompound which is capable of increasing or maintaining the pH of asolution is contacted with the dental surface for about 20 minutes.

Preferably the stabilized ACP and/or ACFP are contacted with the dentalsurface for a period of about 1 minute to 2 hours, or 5 minutes to 60minutes or about 10 minutes. The stabilized ACP and/or ACFP may berepeatedly applied to the dental surface over a period of 1 day toseveral months.

In one embodiment, the compound which is capable of increasing ormaintaining the pH of a solution is contacted with the dental surface 1to 60 minutes, or 1 to 30 minutes, or 1 to 5 minutes prior to contactingthe dental surface with the stabilized ACP and/or ACFP.

In one embodiment, the compound which is capable of increasing ormaintaining the pH of a solution is contacted with the dental surface 1to 60 minutes, or 1 to 30 minutes, or 1 to 5 minutes after contactingthe dental surface with the stabilized ACP and/or ACFP.

In a further aspect of the present invention there is provided a methodfor mineralizing a tooth surface comprising applying a stabilized ACPand/or ACFP complex and a compound which is capable of increasing ormaintaining the pH of a solution to a tooth surface. Preferably thetooth surface is tooth enamel. Typically, the tooth surface is toothenamel containing a lesion selected from the group consisting of one ormore of a white spot lesion; a fluorotic lesion; a caries lesion; or alesion caused by tooth erosion. Preferably, the stabilized ACP and/orACFP complex and a compound which is capable of increasing ormaintaining the pH of a solution is contained in the same compositionthat is applied to the tooth surface. Preferably the compound which iscapable of increasing or maintain the pH of a solution is a base. Thebase may be any base described herein, including, but not limited to,sodium bicarbonate, sodium hypochlorite, a hypofluorite or urea.

In one embodiment, the dental surface is in need of such treatmentTherefore the invention includes in addition to the steps of any methoddescribed herein a step of identifying a subject suffering fluorosis,dental caries, dentinal hypersensitivity or dental calculus, a whitespot lesion; a fluorotic lesion; a caries lesion; or a lesion caused bytooth erosion.

In one aspect, the present invention provides a composition formineralizing a dental surface or subsurface comprising a compound thatis capable of increasing or maintaining the pH of a solution and amineralizing agent. Preferably, the mineralizing agent is stabilizedamorphous calcium phosphate (ACP) and/or amorphous calcium fluoridephosphate (ACFP). Preferably, the compound that is capable of increasingor maintaining the pH of a solution is present in an amount effective toraise the pH of a dental lesion from 6.0 to 7.5. Preferably, thecompound that is capable of increasing or maintaining the pH of asolution is any one or more of the compounds described herein,preferably a base. The base may be any base described herein, including,but not limited to, sodium bicarbonate, sodium hypochlorite, ahypofluorite or urea.

Any composition described herein may be a physiologically acceptablecomposition formulated as a toothpaste, toothpowder, liquid dentifrice,mouthwash, mouthrinse, mouth spray, varnish, dental cement, troche,chewing gum, lozenge, dental paste, gingival massage cream, gargletablet, dairy product and other foodstuffs.

Any composition described herein can be used in any one of the methodsdescribed herein. The composition is a physiologically acceptablecomposition as described herein.

In another aspect, the present invention provides a compositioncomprising a compound that is capable of increasing or maintaining thepH of a solution and a mineralizing agent for use in mineralizing adental surface or subsurface. Preferably, the mineralizing agent isstabilized amorphous calcium phosphate (ACP) and/or amorphous calciumfluoride phosphate (ACFP). Preferably the compound which is capable ofincreasing or maintain the pH of a solution is a base. The base may beany base described herein, including, but not limited to, sodiumbicarbonate, sodium hypochlorite, a hypofluorite or urea.

In a further aspect, there is provided a method of treating orpreventing one or more of each of dental caries, tooth decay, dentalerosion and fluorosis, comprising the steps of administering a compoundcapable of increasing or maintaining the pH of a solution to the teethof a subject followed by administering an ACP or ACFP complex orcomposition. Topical administration of the complex is preferred. Themethod preferably includes the administration of the complex in aformulation as described above. Preferably the compound which is capableof increasing or maintain the pH of a solution is a base. The base maybe any base described herein, including, but not limited to, sodiumbicarbonate, sodium hypochlorite, a hypofluorite or urea.

In a further aspect there is provided the use of a compound capable ofincreasing or maintaining the pH of a solution and a stabilizedamorphous calcium phosphate (ACP) or amorphous calcium fluoridephosphate (ACFP) in the manufacture of a composition for the treatmentand/or prevention of one or more of dental caries, tooth decay, dentalerosion and fluorosis. Preferably the compound which is capable ofincreasing or maintain the pH of a solution is a base. The base may beany base described herein, including, but not limited to, sodiumbicarbonate, sodium hypochlorite, a hypofluorite or urea.

In a further aspect there is provided a composition comprising as anactive agent a compound capable of increasing or maintaining the pH of asolution and a stabilized amorphous calcium phosphate (ACP) or amorphouscalcium fluoride phosphate (ACFP) for mineralizing a dental surface orsubsurface. Typically, mineralizing a dental surface or subsurface isfor the treatment and/or prevention of one or more of dental caries,tooth decay, dental erosion and fluorosis.

In any method or use of the invention there is further provided a stepof applying a source of fluoride ions. The source of fluoride ions maybe applied simultaneously as the compound which is capable of increasingor maintaining the pH of a solution and the source of ACP or ACFP.Alternatively, the source of fluoride ions may be applied prior to, orafter, the compound which is capable of increasing or maintaining the pHof a solution, or the source of ACP or ACFP.

The present invention also provides a composition comprising a compoundthat is capable of increasing or maintaining the pH of a solution and amineralizing agent. Preferably, the mineralizing agent isstabilized-amorphous calcium phosphate (ACP) or amorphous calciumfluoride phosphate (ACFP). Preferably, the composition further includesa pharmaceutically acceptable carrier, diluent or excipient. Preferablythe compound which is capable of increasing or maintaining the pH of asolution is a base. The base may be any base described herein,including, but not limited to, sodium bicarbonate, sodium hypochlorite,a hypofluorite or urea.

In a preferred embodiment of each aspect of the invention, thephosphopeptide stabilized amorphous calcium phosphate (ACP) or amorphouscalcium fluoride phosphate (ACFP) complex in the composition has tightlybound and loosely bound calcium, wherein the bound calcium in thecomplex is less than the tightly bound calcium in an ACP or ACFP complexformed at a pH of 7.0. Optionally, the ACP or ACFP is predominantly in abasic form.

In another preferred embodiment of each aspect of the invention, thecalcium ion content of the stabilized ACP or ACFP complex in thecomposition is in the range of about 30 to 100 moles of calcium per moleof PP. More preferably, the calcium ion content is in the range of about30 to about 50 moles of calcium per mole of PP.

In any embodiment, the ACP and/or ACFP in the composition can be in theform of a casein phosphopeptide stabilized ACP and/or ACFP complex.

In another aspect the invention is a physiologically acceptablecomposition including stabilized amorphous calcium phosphate (ACP)and/or amorphous calcium fluoride phosphate (ACFP) as described herein,a base and an excipient, diluent or carrier suitable for oraladministration. The base may be any base described herein, including,but not limited to, sodium bicarbonate, sodium hypochlorite, ahypofluorite or urea.

The invention also relates to a kit for the treatment or prevention ofone or more of dental caries, fluorosis and dental erosion including (a)a compound capable of increasing or maintaining the pH of a solution and(b) a stabilized-ACP and/or stabilized-ACFP complex in apharmaceutically acceptable carrier. Desirably, the kit further includesinstructions for their use for the mineralization of a dental surface ina patient in need of such treatment. The instructions may describe theuse of the kit to treat or prevent one or more of each of dental caries,tooth decay, dental erosion and fluorosis. In one embodiment, the agentand the complex are present in suitable amounts for treatment of apatient. Preferably, the stabilized ACP and/or ACFP is phosphopeptide(PP)-stabilized. Preferably, the phosphopeptide (as defined below) is acasein phosphopeptide. Preferably, the ACP or ACFP is in the form of acasein phosphopeptide stabilized ACP or ACFP complex.

The composition or kit of the invention may further include a source offluoride ions. The fluoride ions may be from any suitable source. Asource of fluoride ions may include free fluoride ions or fluoridesalts. Examples of sources of fluoride ions include, but are not limitedto the following: sodium fluoride, sodium monofluorophosphate, stannousfluoride, sodium silicofluoride, silver fluoride, amine fluoride or anymetal ion fluoride salt. A source of fluoride ions may be ahypofluorite. These sources of fluoride ions may be provided in solution(typically an aqueous solution), or a suspension.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Further aspects of the present invention and further embodiments of theaspects described in the preceding paragraphs will become apparent fromthe following description, given by way of example and with reference tothe accompanying drawings. It will be understood that the inventiondisclosed and defined in this specification extends to all alternativecombinations of two or more of the individual features mentioned orevident from the text or drawings. All of these different combinationsconstitute various alternative aspects of the invention.

Reference will now be made in detail to certain embodiments of theinvention. While the invention will be described in conjunction with theembodiments, it will be understood that the intention is not to limitthe invention to those embodiments. On the contrary, the invention isintended to cover all alternatives, modifications, and equivalents,which may be included within the scope of the present invention asdefined by the claims.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. The present invention is in no waylimited to the methods and materials described.

All of the patents and publications referred to herein are incorporatedby reference in their entirety.

For purposes of interpreting this specification, terms used in thesingular will also include the plural and vice versa.

As used herein, except where the context requires otherwise, the term“comprise” and variations of the term, such as “comprising”, “comprises”and “comprised”, are not intended to exclude further additives,components, integers or steps. As used herein, except where the contextrequires otherwise, “comprise” and “include” can be usedinterchangeably.

In one aspect, the present invention provides a method of mineralizing adental surface or subsurface comprising contacting the dental surface orsubsurface with a compound that is capable of increasing or maintainingthe pH of a solution and a mineralizing agent. A dental subsurface istypically a hypomineralized lesion such that the compound andmineralizing agent contacted to the dental surface migrates through anysurface layer, i.e. pellicle and/or plaque, through the porous dentalsurface to the region requiring mineralization. Preferably, themineralizing agent is stabilized amorphous calcium phosphate (ACP)and/or amorphous calcium fluoride phosphate (ACFP). The dental surfaceis preferably dental enamel. The dental surface may be a lesion in theenamel, such as a lesion caused by caries, dental erosion or fluorosis.

Any compound that is capable of increasing or maintaining the pH of asolution can be used in the method of the present invention. Withoutbeing bound by any theory or mode of action it is believed that themaintenance of a basic pH during intra-lesion mineralization minimisesany restriction on the mineralization process by any acidic molecularspecies. Therefore, it is believed that the compound which is capable ofincreasing or maintaining the pH of a solution promotes mineralizationby neutralising acid produced during the mineralization process.

Mineralization of dental surfaces can be significantly enhanced byincreasing the pH of a lesion during the process of mineralization. Inparticular, it has been found that the mineralization of enamel bystabilized soluble forms of ACP (CPP-ACP) and ACFP (CPP-ACFP) isenhanced by a compound that increases the intra-lesion pH if theintra-lesion pH is acidic or maintains the intra-lesion pH if theintra-lesion pH is neutral or basic. For example, during the developmentof caries, the pH of the intra-lesion fluid may be 6 or below. Thecompound one that can raise, or maintain, the pH at whichremineralization of a hypomineralised surface or subsurface can occur.

The compound which is capable of increasing or maintaining the pH of asolution may be contacted with the dental surface for a period of about1 to 60 minutes, or for about 1 to 30 minutes. In one embodiment, thecompound which is capable of increasing or maintaining the pH of asolution is contacted with the dental surface for about 20 minutes. Anexample of how this is achieved is formulating the compound into an oralcomposition, such as a paste, and then contacting or applying thecomposition to the dental surface. The oral composition, such as apaste, has sufficient viscosity to be retained on the tooth for therequired time period.

Preferably the stabilized ACP and/or ACFP is contacted with the dentalsurface for a period of about 1 minute to 2 hours, or 5 minutes to 60minutes or about 10 minutes. The stabilized ACP and/or ACFP may berepeatedly applied to the dental surface over a period of 1 day toseveral months.

In one embodiment, the compound which is capable of increasing ormaintaining the pH of a solution is contacted with the dental surface 1to 60 minutes, or 1 to 30 minutes, or 1 to 5 minutes prior to contactingthe dental surface with the stabilized ACP and/or ACFP.

In one embodiment, the compound which is capable of increasing ormaintaining the pH of a solution is contacted with the dental surface 1to 60 minutes, or 1 to 30 minutes, or 1 to 5 minutes after contactingthe dental surface with the stabilized ACP and/or ACFP.

In a further aspect of the present invention there is provided a methodfor mineralizing a tooth surface comprising applying an ACP and/or ACFPcomplex and a compound which is capable of increasing or maintaining thepH of a solution to a tooth surface. Preferably the tooth surface istooth enamel. Typically, the tooth surface is tooth enamel containing alesion selected from the group consisting of one or more of a white spotlesion; a fluorotic lesion; a caries lesion; or a lesion caused by tootherosion.

In one embodiment, the dental surface is in need of such treatment.Therefore, in another aspect, the invention includes in addition to thesteps of any method described herein a step of identifying a subjectsuffering fluorosis, dental caries, dentinal hypersensitivity or dentalcalculus, a white spot lesion; a fluorotic lesion; a caries lesion; or alesion caused by tooth erosion.

In another aspect, the present invention provides a compositioncomprising a compound that is capable of increasing or maintaining thepH of a solution and a mineralizing agent for use in mineralizing adental surface or subsurface.

A compound which is capable of increasing or maintaining the pH of asolution includes a compound which can accept hydrogen cations (protons)or, more generally, donate a pair of valence electrons. Preferably, thecompound may commonly be a base. The compound is capable of increasingthe pH of a solution that has an acidic pH (i.e. less than pH 7).Preferably, the compound is capable of raising the pH of intra-lesionfluid of a dental lesion from 6 to 7.5. In one embodiment, a compoundwhich is capable of increasing or maintaining the pH of a solution is analkali which has the capacity to release hydroxide ions.

A compound which is capable of increasing or maintaining the pH of asolution also includes a compound that can maintain as a buffer the pHof a neutral or basic solution (i.e. pH greater than or equal to 7) whenthe neutral or basic solution is exposed to an acid. Typically, thecompound is capable of maintaining the pH of a solution between 7 to 9,preferably about 7.5. As used herein reference to the increasing ormaintaining the pH of a solution includes increasing or maintaining thepH the fluid in a subsurface lesion, i.e. intra-lesion fluid.

Any pharmaceutically acceptable compounds described as a base aresuitable for use in the invention. Typically, the base is suitable fororal use. Preferably, the compound acts as a base, i.e. only releaseshydroxide ions or donates electrons, in the presence of an acid. Thebase may be a free-base form, or in a pharmaceutically acceptable saltform. Non-limiting examples of bases suitable for use in the inventioninclude hydroxides, chlorides, borates, phosphates including hydrogenphosphates and dihydrogen phosphates, citrates, carbonates,bicarbonates, hypochlorites, amines and any salt forms thereof includingan alkali metal salt forms. More specifically, non-limiting examples ofsuitable pharmaceutically acceptable bases include ammonium hydroxide,sodium hydroxide, potassium hydroxide, lithium hydroxide, calciumhydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide,copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine,ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine,arginine, histidine. A hypofluorite capable of acting as a base asdescribed herein is also useful in the invention as the agent forincreasing or maintaining pH. A suitable hypofluorite would react insitu to produce fluoride ions and hydroxide (or another base) ions. Oneskilled in the art will appreciate that fluoride ions can substitute forhydroxide in the crystal structure of apatite forming fluorapatite.

A stabilized-ACP or ACFP complex as described in the currentspecification may be the “closed” complexes are shown in FIG. 2 of Crosset al., 2007 Current Pharmaceutical Design, 13, 793-800.

A stabilized-ACP or ACFP complex as referred to herein includes astabilized-ACP or ACFP complex as described in WO2006/056013 thecontents of which are incorporated by reference.

In a preferred embodiment, the phosphopeptide stabilized amorphouscalcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP)complex has tightly bound and loosely bound calcium, wherein the boundcalcium in the complex is less than the tightly bound calcium in an ACPor ACFP complex formed at a pH of 7.0. Optionally, the ACP or ACFP ispredominantly in a basic form.

A stabilized-ACP or ACFP complex as referred to herein include astabilized-ACP or ACFP complex formed at a pH of below 7.0. Preferablythe complex is formed at a pH in the range of about 5.0 up to but below7.0. More preferably the complex is formed at a pH range of about 5.0 toabout 6.0. In a preferred embodiment, the complex is formed at a pH ofabout 5.0 or about 5.5. Preferably, the ACP or ACFP in the complex ispredominantly in a basic form.

A stabilized-ACP may be produced by a method comprising the steps of:

-   (i) obtaining a solution comprising at least one phosphopeptide and;-   (ii) admixing solutions comprising calcium ions, phosphate ions and    hydroxide ions, while maintaining the pH at about 7.0 or below.

A stabilized ACFP may be produced by a method comprising the steps of:

-   (i) obtaining a solution comprising at least one phosphopeptide and;-   (ii) admixing solutions comprising calcium ions, phosphate ions,    hydroxide ions and fluoride ions, while maintaining the pH at about    7.0 or below.

The hydroxide ions may be titrated into the solution to maintain thephosphopeptide solution at an essentially constant pH. The calcium andphosphate ions may be titrated into the phosphopeptide solution withconstant mixing and at a rate that avoids the formation of a calciumphosphate precipitate in the phosphopeptide solution.

A phosphopeptide stabilized amorphous calcium phosphate (ACP) oramorphous calcium fluoride phosphate (ACFP) complex may also includewherein the ACP in the complex has tightly bound and loosely boundcalcium, wherein the tightly bound calcium in the complex is less thanthe tightly bound calcium in an ACP or ACFP complex formed at a pH of7.0 and the ACP or ACFP is predominantly in a basic form, obtainable orobtained by a method comprising:

-   -   a) admixing a first solution comprising calcium ions, a second        solution comprising phosphate ions, and optionally a third        solution comprising fluoride ions, to a solution comprising        phosphopeptides and a solvent with a pH of from about 5 up to        but below 7; and    -   b) maintaining the pH of the solution at about 5.0 up to but        below 7.0 during the admixing by adding hydroxide ions.

“Tightly” and “loosely” bound calcium and phosphate can be determinedusing analytical ultrafiltration as shown in Example 2. Briefly, thesolution of phosphopeptide, calcium, phosphate and optionally fluorideadmixed while maintaining the pH at about 7.0 or below can be firstfiltered through a 0.1 micron filter to remove free calcium andphosphate that is not associated with the complexes. This free calciumand phosphate is present in the filtrate and discarded. Any free calciumor phosphate that is not associated in any way with the complexes wouldnot be bioavailable, i.e. delivered by the phosphopeptide to the tooth.The retentate from the 0.1 micron filtration can be further analyzed bycentrifugation through a 3000 mw cutoff filter at 1,000 g for 15 min.The resulting filtrate contains calcium and phosphate that is looselybound or associated with the complexes. At this centrifugal forcecalcium and phosphate that is not tightly bound to the complexes arereleased and move into the filtrate. The calcium and phosphate that istightly bound in the complexes is retained in the retentate. The amountof tightly bound calcium and phosphate in the retentate can then bedetermined by subtracting the amount of calcium and phosphate in thefiltrate from the total amount of calcium and phosphate in the retentateof the 0.1 micron filtration.

A stabilized-ACP or ACFP complex as referred to herein include astabilized-ACP or ACFP complex as described in WO2006/135982 thecontents of which are incorporated by reference.

A “superloaded” phosphopeptide or phosphoprotein (PP) is aphosphopeptide or phosphoprotein that has been superloaded with calciumand phosphate ions. The calcium ions of the superloaded PP may be in therange 30-1000 mol Ca per mole of PP, or in the range of 30-100 or 30-50mole Ca per mole of PP. In another embodiment, the mol Ca per mol of PPis at least 25, 30, 35, 40, 45 or 50. It can be a stabilized-amorphouscalcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP)complex. The complex may be formed at any pH (e.g. 3-10). Preferably thephosphopeptide includes the sequence -A-B-C-, where A is a phosphoaminoacid, preferably phosphoserine, B is any amino acid including aphosphoamino acid and C is glutamic acid, aspartic acid or aphosphoamino acid. The phosphoamino acid may be phosphoserine.

In one aspect, the present invention includes a phosphopeptide orphosphoprotein (PP) stabilized amorphous calcium phosphate or amorphouscalcium fluoride phosphate complex having a calcium ion content greaterthan about 30 moles of calcium per mole of PP. In a preferredembodiment, the calcium ion content is in the range of about 30 to 100moles of calcium per mole of PP. More preferably, the calcium ioncontent is in the range of about 30 to about 50 moles of calcium permole of PP.

The invention also provides a phosphopeptide or phosphoprotein (PP)stabilized-amorphous calcium phosphate (ACP) or amorphous calciumfluoride phosphate (ACFP) complex produced by a method comprising thesteps of.

-   (i) obtaining solutions comprising calcium, inorganic phosphate and    fluoride (optional); and-   (ii) admixing (i) with a solution comprising PP-ACP.

In a preferred embodiment, the PP is casein phosphopeptide (CPP).

In a further aspect, the present invention also includes use of aformulation of a PP stabilized ACP and/or ACFP complex together with atleast an equal amount by weight of calcium phosphate. Preferably thecalcium phosphate is CaHPO₄ or calcium lactate or any other solublecalcium phosphate compound. Preferably, the calcium phosphate (e.g.CaHPO₄) is dry blended with the PP stabilized ACP and/or ACFP complex.In a preferred embodiment, the PP-ACP and/or PP-ACFP complex:calciumphosphate ratio is about 1:1-50, more preferably about 1:1-25, morepreferably about 1:5-15. In one embodiment, the PP-ACP and/or PP-ACFPcomplex:calcium phosphate ratio is about 1:10.

The oral care formulation that includes a phosphopeptide orphosphoprotein (PP) stabilized amorphous calcium phosphate (ACP) and/oramorphous calcium fluoride phosphate (ACFP) complex having a calcium ioncontent greater than about 30 moles of calcium per mole of PP when usedin the oral cavity may be produced by a method including the steps of:

-   (i) obtaining a powder including a PP-ACP and/or PP-ACFP complex;-   (ii) dry blending with an effective amount of calcium phosphate; and-   (iii) formulating the dry blended PP-ACP and/or PP-ACFP and calcium    phosphate mixture into an oral care formulation.

Preferably, the form of calcium phosphate for dry blending is anysoluble calcium phosphate including, but not limited to, CaHPO₄, Ca₂HPO₄and calcium lactate.

The present invention also provides a method of mineralizing a dentalsurface or subsurface including the steps of:

-   (i) contacting the dental surface with a protein disrupting agent,    and-   (ii) contacting the dental surface with a composition of the    invention.

The dental surface is preferably dental enamel. In one embodiment thedental surface is a lesion in the enamel, such as a lesion caused bycaries, dental erosion or fluorosis. Any suitable protein disruptingagent can be used in the method of the present invention. The agent isrequired to reduce the proteinaceous barrier formed over the surface tobe treated, such as the pellicle over teeth. Examples of suitable agentsinclude bleach, detergent, chaotropic agents such as urea, highphosphate concentrations, cocktails of proteases (e.g. endopeptidases,proteinases and exopeptidases) and any other protein solubilizing,disrupting or hydrolysing agent. Examples of suitable bleaches includesodium hypochlorite (NaOCl), and carbamide peroxide bleaches. In apreferred embodiment, the bleach is an alkaline bleach. In a furtherpreferred embodiment the alkaline bleach is NaOCl. The proteindisrupting agent acts to solubilize and partially or wholly removeproteins from the dental surface, particularly proteins of the pellicle.

A composition as described herein may further include free fluorideions. The fluoride ions may be from any suitable source. A source offluoride ions may include free fluoride ions or fluoride salts. Examplesof sources of fluoride ions include, but are not limited to thefollowing: sodium fluoride, sodium monofluorophosphate, stannousfluoride, sodium silicofluoride and amine fluoride. These may beprovided in solution (typically an aqueous solution), or a suspension.

The fluoride ions are preferably present in the composition in an amountgreater than 1 ppm. More preferably, the amount is more than 3 ppm. Inanother embodiment, it is preferably more than 10 ppm. In typicalembodiments described below, the amount may be several hundred orthousand ppm. The fluoride content is typically measured as a ppm inoral compositions in the manner commonly used in the art. Where thefluoride is provided from a source with the stabilized ACP, the ppmrefers to the concentration of the fluoride in that source, typically asolution or suspension of bioavailable fluoride.

In any aspect or embodiments as described herein, the stabilized ACPand/or ACFP is phosphopeptide (PP)-stabilized. Preferably, thephosphopeptide (as defined below) is a casein phosphopeptide.Preferably, the ACP or ACFP is in the form of a casein phosphopeptidestabilized ACP or ACFP complex.

“Phosphopeptide” in the context of the description of this inventionmeans an amino acid sequence in which at least one amino acid isphosphorylated. Preferably, the phosphopeptide includes one or more ofthe amino acid sequence -A-B-C-, where A is a phosphoamino residue, B isany amino acyl residue including a phosphoamino residue and C isselected from a glutamyl, aspartyl or phosphoamino residue. Any of thephosphoamino residues may independently be a phosphoseryl residue. B isdesirably a residue the side-chain of which is neither relatively largenor hydrophobic. It may be Gly, Ala, Val, Met, Leu, lie, Ser, Thr, Cys,Asp, Glu, Asn, Gin or Lys. Preferably at least two of the phosphoaminoacids in the sequence are preferably contiguous. Preferably thephosphopeptide includes the sequence A-B-C-D-E, where A, B, C, D and Eare independently phosphoserine, phosphothreonine, phosphotyrosine,phosphohistidine, glutamic acid or aspartic acid, and at least two,preferably three, of the A, B, C, D and E are a phosphoamino acid. In apreferred embodiment, the phosphoamino acid residues are phosphoserine,most preferably three contiguous phosphoserine residues. It is alsopreferred that D and E are independently glutamic or aspartic acid.

In one embodiment, the ACP or ACFP is stabilized by a caseinphosphopeptide (CPP), which is in the form of intact casein or fragmentof the casein, and the complex formed preferably has the formula[CPP(ACP)_(8]n) or [(CPP)(ACFP)₈]n where n is equal to or greater than1, for example 6. The complex formed may be a colloidal complex, wherethe core particles aggregate to form large (e.g. 100 nm) colloidalparticles suspended in water. Thus, the PP can be a casein protein or aphosphopeptide.

The PP may be from any source; it may be present in the context of alarger polypeptide, including a full length casein polypeptide, or itmay be isolated by tryptic or other enzymatic or chemical digestion ofcasein, or other phosphoamino acid rich proteins such as phosphitin, orby chemical or recombinant synthesis, provided that it comprises thesequence -A-B-C- or A-B-C-D-E as described above. The sequence flankingthis core sequence may be any sequence. However, those flankingsequences in α_(s1)(59-79), _(β)(1-25), α_(s2)(46-70) and α_(s2)(1-21)are preferred. The flanking sequences may optionally be modified bydeletion, addition or conservative substitution of one or more residues.The amino acid composition and sequence of the flanking region are notcritical.

The phosphopeptide may be selected from any described in WO2006/056013,WO2006/135982 or U.S. Pat. No. 5,015,628.

Examples of conservative substitutions are shown in Table 1 below.

TABLE 1 Exemplary Conservative Preferred Conservative Original ResidueSubstitution Substitution Ala Val, Leu, Ile Val Asn Gln Lys His Phe GlnGln Asn Asn Gly Pro Pro Ile Leu, Val, Met, Ala, Phe Leu Leu Ile, Val,Met, Ala, Phe Ile Lys Arg, Gln, Asn Arg Phe Leu, Val, Ile, Ala Leu ProGly Gly Ser Thr Thr Val Ile, Leu, Met, Phe, Ala Leu Asp Glu Glu Thr SerSer Trp Tyr Tyr Tyr Trp Phe Thr Ser Phe

The flanking sequences may also include non-naturally occurring aminoacid residues. Commonly encountered amino acids which are not encoded bythe genetic code, include:

-   -   2-amino adipic acid (Aad) for Glu and Asp;    -   2-aminopimelic acid (Apm) for Glu and Asp;    -   2-aminobutyric (Abu) acid for Met, Lou, and other aliphatic        amino acids;    -   2-aminoheptanoic acid (Ahe) for Met, Leu and other aliphatic        amino acids;    -   2-aminoisobutyric acid (Aib) for Gly;    -   cyclohexylalanine (Cha) for Val, and Leu and lie;    -   homoarginine (Har) for Arg and Lys;    -   2,3-diaminopropionic acid (Dpr) for Lys, Arg and His;    -   N-ethylglycine (EtGly) for Gly, Pro, and Ala;    -   N-ethylasparigine (EtAsn) for Asn, and Gin;    -   Hydroxyllysine (Hyl) for Lys;    -   allohydroxyllysine (AHyl) for Lys;    -   3-(and 4) hydroxyproline (3Hyp, 4Hyp) for Pro, Ser, and Thr;    -   alloisoleucine (Alle) for lle, Leu, and Val;    -   ρ-amidinophenylalanine for Ala;    -   N-methylglycine (MeGly, sarcosine) for Gly, Pro, Ala.    -   N-methylisoleucine (Melle) for lle;    -   Norvaline (Nva) for Met and other aliphatic amino acids;    -   Norleucine (Nle) for Met and other aliphatic amino acids;    -   Omithine (Orn) for Lys, Arg and His;    -   Citrulline (Cit) and methionine sulfoxide (MSO) for Thr, Asn and        Gln;    -   N-methylphenylalanine (MePhe), trimethylphenylalanine, halo (F,        Cl, Br and I) phenylalanine, triflourylphenylalanine, for Phe.

In one embodiment, the PP is one or more phosphopeptides selected fromthe group consisting of α_(s1)(59-79), β(1-25), α_(s2)(46-70) andα_(s2)(1-21).

In another embodiment of the invention, the stabilized ACFP or ACPcomplex and a compound capable of increasing or maintaining the pH of asolution is incorporated into oral compositions such as toothpaste,mouth washes or formulations for the mouth to aid in the preventionand/or treatment of dental caries, tooth decay, dental erosion orfluorosis. The ACFP or ACP complex may comprise 0.01-50% by weight ofthe composition, preferably 1.0-50%. For oral compositions, it ispreferred that the amount of the CPP-ACP and/or CPP-ACFP administered is0.01-50% by weight, preferably 1.0%-50% by weight of the composition. Ina particularly preferred embodiment, the oral composition of the presentinvention contains about 2% CPP-ACP, CPP-ACFP or a mixture of both. Theoral composition of this invention which contains the above-mentionedagents may be prepared and used in various forms applicable to the mouthsuch as dentifrice including toothpastes, toothpowders and liquiddentifrices, mouthwashes, mouth rinses, mouth sprays, varnish, dentalcement, troches, chewing gums, dental pastes, gingival massage creams,gargle tablets, dairy products and other foodstuffs. The oralcomposition according to this invention may further include additionalwell known ingredients depending on the type and form of a particularoral composition.

In certain preferred forms of the invention the oral composition may besubstantially liquid in character, such as a mouthwash, rinse or spray.In such a preparation the vehicle is typically a water-alcohol mixturedesirably including a humectant as described below. Generally, theweight ratio of water to alcohol is in the range of from about 1:1 toabout 20:1. The total amount of water-alcohol mixture in this type ofpreparation is typically in the range of from about 70 to about 99.9% byweight of the preparation. The alcohol is typically ethanol orisopropanol. Ethanol is preferred.

In other desirable forms of this invention, the composition may besubstantially solid or pasty in character, such as toothpowder, a dentaltablet or a toothpaste (dental cream) or gel dentifrice. The vehicle ofsuch solid or pasty oral preparations generally contains dentallyacceptable polishing material. Examples of polishing materials arewater-insoluble sodium metaphosphate, potassium metaphosphate,tricalcium phosphate, dihydrated calcium phosphate, anhydrous dicalciumphosphate, calcium pyrophosphate, magnesium orthophosphate, trimagnesiumphosphate, calcium carbonate, hydrated alumina, calcined alumina,aluminium silicate, zirconium silicate, silica, bentonite, and mixturesthereof. Other suitable polishing material include the particulatethermosetting resins such as melamine-, phenolic, andurea-formaldehydes, and cross-linked polyepoxides and polyesters.Preferred polishing materials include crystalline silica having particlesizes of up to about 5 microns, a mean particle size of up to about 1.1microns, and a surface area of up to about 50,000 cm²/g., silica gel orcolloidal silica, and complex amorphous alkali metal aluminosilicate.

When visually clear gels are employed, a polishing agent of colloidalsilica, such as those sold under the trademark SYLOID as Syloid 72 andSyloid 74 or under the trademark SANTOCEL as Santocel 100, alkali metalaluminosilicate complexes are particularly useful since they haverefractive indices close to the refractive indices of gellingagent-liquid (including water and/or humectant) systems commonly used indentifrices.

Many of the so-called “water insoluble” polishing materials are anionicin character and also include small amounts of soluble material. Thus,insoluble sodium metaphosphate may be formed in any suitable manner, forexample as illustrated by Thorpe's Dictionary of Applied Chemistry,Volume 9, 4th Edition, pp. 510-511. The forms of insoluble sodiummetaphosphate known as Madrell's salt and Kurrol's salt are furtherexamples of suitable materials. These metaphosphate salts exhibit only aminute solubility in water, and therefore are commonly referred to asinsoluble metaphosphates (IMP). There is present therein a minor amountof soluble phosphate material as impurities, usually a few percent suchas up to 4% by weight. The amount of soluble phosphate material, whichis believed to include a soluble sodium trimetaphosphate in the case ofinsoluble metaphosphate, may be reduced or eliminated by washing withwater if desired. The insoluble alkali metal metaphosphate is typicallyemployed in powder form of a particle size such that no more than 1% ofthe material is larger than 37 microns.

The polishing material is generally present in the solid or pastycompositions in weight concentrations of about 10% to about 99%.Preferably, it is present in amounts from about 10% to about 75% intoothpaste, and from about 70% to about 99% in toothpowder. Intoothpastes, when the polishing material is silicious in nature, it isgenerally present in an amount of about 10-30% by weight. Otherpolishing materials are typically present in amount of about 30-75% byweight.

In a toothpaste, the liquid vehicle may comprise water and humectanttypically in an amount ranging from about 10% to about 80% by weight ofthe preparation. Glycerine, propylene glycol, sorbitol and polypropyleneglycol exemplify suitable humectants/carriers. Also advantageous areliquid mixtures of water, glycerine and sorbitol. In clear gels wherethe refractive index is an important consideration, about 2.5-30% w/w ofwater, 0 to about 70% w/w of glycerine and about 20-80% w/w of sorbitolare preferably employed.

Toothpaste, creams and gels typically contain a natural or syntheticthickener or gelling agent in proportions of about 0.1 to about 10,preferably about 0.5 to about 5% w/w. A suitable thickener is synthetichectorite, a synthetic colloidal magnesium alkali metal silicate complexclay available for example as Laponite (e.g. CP, SP 2002, D) marketed byLaporte Industries Limited. Laponite D is, approximately by weight58.00% SiO₂, 25.40% MgO, 3.05% Na₂O, 0.98% Li₂O, and some water andtrace metals. Its true specific gravity is 2.53 and it has an apparentbulk density of 1.0 g/ml at 8% moisture.

Other suitable thickeners include Irish moss, iota carrageenan, gumtragacanth, starch, polyvinylpyrrolidone, hydroxyethylpropylcellulose,hydroxybutyl methyl cellulose, hydroxypropyl methyl cellulose,hydroxyethyl cellulose (e.g. available as Natrosol), sodiumcarboxymethyl cellulose, and colloidal silica such as finely groundSyloid (e.g. 244). Solubilizing agents may also be included such ashumectant polyols such propylene glycol, dipropylene glycol and hexyleneglycol, cellosolves such as methyl cellosolve and ethyl cellosolve,vegetable oils and waxes containing at least about 12 carbons in astraight chain such as olive oil, castor oil and petrolatum and esterssuch as amyl acetate, ethyl acetate and benzyl benzoate.

It will be understood that, as is conventional, the oral preparationswill usually be sold or otherwise distributed in suitable labelledpackages. Thus, a jar of mouth rinse will have a label describing it, insubstance, as a mouth rinse or mouthwash and having directions for itsuse; and a toothpaste, cream or gel will usually be in a collapsibletube, typically aluminium, lined lead or plastic, or other squeeze, pumpor pressurized dispenser for metering out the contents, having a labeldescribing it, in substance, as a toothpaste, gel or dental cream.

Organic surface-active agents may be used in the compositions of thepresent invention to achieve increased prophylactic action, assist inachieving thorough and complete dispersion of the active agentthroughout the oral cavity, and render the instant compositions morecosmetically acceptable. The organic surface-active material ispreferably anionic, non-ionic or ampholytic in nature and preferablydoes not interact with the active agent. It is preferred to employ asthe surface-active agent a detersive material which imparts to thecomposition detersive and foaming properties. Suitable examples ofanionic surfactants are water-soluble salts of higher fatty acidmonoglyceride monosulfates, such as the sodium salt of the monosulfatedmonoglyceride of hydrogenated coconut oil fatty acids, higher alkylsulfates such as sodium lauryl sulfate, alkyl aryl sulfonates such assodium dodecyl benzene sulfonate, higher alkylsulfo-acetates, higherfatty acid esters of 1,2-dihydroxy propane sulfonate, and thesubstantially saturated higher aliphatic acyl amides of lower aliphaticamino carboxylic acid compounds, such as those having 12 to 16 carbonsin the fatty acid, alkyl or acyl radicals, and the like. Examples of thelast mentioned amides are N-lauroyl sarcosine, and the sodium,potassium, and ethanolamine salts of N-lauroyl, N-myristoyl, orN-palmitoyl sarcosine which should be substantially free from soap orsimilar higher fatty acid material. The use of these sarconite compoundsin the oral compositions of the present invention is particularlyadvantageous since these materials exhibit a prolonged marked effect inthe inhibition of acid formation in the oral cavity due to carbohydratesbreakdown in addition to exerting some reduction in the solubility oftooth enamel in acid solutions. Examples of water-soluble non-ionicsurfactants suitable for use are condensation products of ethylene oxidewith various reactive hydrogen-containing compounds reactive therewithhaving long hydrophobic chains (e.g. aliphatic chains of about 12 to 20carbon atoms), which condensation products (“ethoxamers”) containhydrophilic polyoxyethylene moieties, such as condensation products ofpoly (ethylene oxide) with fatty acids, fatty alcohols, fatty amides,polyhydric alcohols (e.g. sorbitan monostearate) and polypropyleneoxide(e.g. Pluronic materials).

The surface active agent is typically present in amount of about 0.1-5%by weight. It is noteworthy, that the surface active agent may assist inthe dissolving of the active agent of the invention and thereby diminishthe amount of solubilizing humectant needed.

Various other materials may be incorporated in the oral preparations ofthis invention such as whitening agents, preservatives, silicones,chlorophyll compounds and/or ammoniated material such as urea,diammonium phosphate, and mixtures thereof. These adjuvants, wherepresent, are incorporated in the preparations in amounts which do notsubstantially adversely affect the properties and characteristicsdesired.

Any suitable flavouring or sweetening material may also be employed.Examples of suitable flavouring constituents are flavouring oils, e.g.oil of spearmint, peppermint, wintergreen, sassafras, clove, sage,eucalyptus, marjoram, cinnamon, lemon, and orange, and methylsalicylate. Suitable sweetening agents include sucrose, lactose,maltose, sorbitol, xylitol, sodium cyclamate, perillartine, AMP(aspartyl phenyl alanine, methyl ester), saccharine, and the like.Suitably, flavour and sweetening agents may each or together comprisefrom about 0.1% to 5% more of the preparation.

The compositions of this invention can also be incorporated in lozenges,or in chewing gum or other products, e.g. by stirring into a warm gumbase or coating the outer surface of a gum base, illustrative of whichare jelutong, rubber latex, vinylite resins, etc., desirably withconventional plasticizers or softeners, sugar or other sweeteners orsuch as glucose, sorbitol and the like. The composition of the inventionmay be a dual phase composition wherein each phase permits release ofcomponents over different time periods. For example, in use a dual phasecomposition may release stabilized ACP and/or ACFP, preferably CPP-ACPand/or CPP-ACFP, from a first phase at a faster rate than a compoundthat is capable of increasing or maintaining the pH of a solution from asecond phase. Preferably, the dual phase composition is a dual phasechewing gum.

In a further aspect, the invention provides compositions includingpharmaceutical compositions comprising any of the ACFP and/or ACPcomplexes as described above together with a compound capable ofincreasing or maintaining the pH of a solution and apharmaceutically-acceptable carrier. Such compositions may be selectedfrom the group consisting of dental, anticariogenic compositions andtherapeutic compositions. Dental compositions or therapeuticcompositions may be in the form of a gel, liquid, solid, powder, creamor lozenge. Therapeutic compositions may also be in the form of tabletsor capsules. In one embodiment, the ACP and/or ACFP complexes aresubstantially the only remineralizing active components of such acomposition. For example, a crème formulation may be employedcontaining: water; glycerol; CPP-ACP; D-sorbitol; silicon dioxide;sodium carboxymethylcellulose (CMC-Na); propylene glycol; titaniumdioxide; xylitol; phosphoric acid; guar gum; zinc oxide; sodiumsaccharin; ethyl p-hydroxybenzoate; magnesium oxide; butyl phydroxybenzoate and propyl p-hydroxybenzoate.

The invention further includes a formulation described above providedtogether with instructions for its use to treat or prevent any one ormore of dental caries or tooth decay, dental erosion and fluorosis.

In one embodiment, the active components of the composition consistessentially of the compound capable of increasing or maintaining the pHof a solution and stabilized ACP and/or ACFP. It is believed, withoutbeing bound by any theory or mode of action, that the stabilized ACPand/or ACFP and the compound capable of increasing or maintaining the pHof a solution are central to the therapeutic or preventative effect ofthe above embodiments of the invention, and thus embodiments consistingessentially of those components (with carriers, excipients and the likeas required) are included within the scope of the invention.

The invention also relates to a kit for the treatment or prevention ofone or more of dental caries, fluorosis and dental erosion including (a)a compound capable of increasing or maintaining the pH of a solution and(b) a CPP-ACP or CPP-ACFP complex in a pharmaceutically acceptablecarrier. Desirably, the kit further includes instructions for their usefor the mineralization of a dental surface in a patient in need of suchtreatment. The instructions may describe the use of the kit to treat orprevent one or more of each of dental caries, tooth decay, dentalerosion and fluorosis. In one embodiment, the agent and the complex arepresent in suitable amounts for treatment of a patient. The instructionsmay direct the user to apply the a compound capable of increasing ormaintaining the pH of a solution before, simultaneously or after aCPP-ACP or CPP-ACFP complex in a pharmaceutically acceptable carrier.

In another aspect a kit of the invention is constructed so that acompound capable of increasing or maintaining the pH of a solution isdispensed simultaneously or after a CPP-ACP or CPP-ACFP complex in apharmaceutically acceptable carrier.

In a further aspect, there is provided a method of treating orpreventing one or more of each of dental caries, tooth decay, dentalerosion and fluorosis, comprising the steps of administering a compoundcapable of increasing or maintaining the pH of a solution to the teethof a subject followed by administering an ACP or ACFP complex orcomposition. Topical administration of the complex is preferred. Themethod preferably includes the administration of the complex in aformulation as described above.

In a further aspect there is provided the use of a compound capable ofincreasing or maintaining the pH of a solution and a stabilizedamorphous calcium phosphate (ACP) or amorphous calcium fluoridephosphate (ACFP) in a manufacture of a composition for the treatmentand/or prevention of one or more of dental caries, tooth decay, dentalerosion and fluorosis.

In a further aspect there is provided a composition comprising as anactive agent a compound capable of increasing or maintaining the pH of asolution and a stabilized amorphous calcium phosphate (ACP) or amorphouscalcium fluoride phosphate (ACFP) for mineralizing a dental surface orsubsurface. Typically, mineralizing a dental surface or subsurface isfor the treatment and/or prevention of one or more of dental caries,tooth decay, dental erosion and fluorosis.

According to a further aspect of the invention there is provided acomposition for dental restoration, including a dental restorativematerial to which has been added a composition of the invention. Thebase of the dental restorative material can be a glass ionomer cement, acomposite material or any other restorative material which iscompatible. It is preferred that the amount of stabilized ACP or ACFP,preferably CPP-ACP complex or CPP-ACFP complex, included in the dentalrestorative material is 0.01-80% by weight, preferably 0.5 10% and morepreferably 1-5% by weight. The dental restorative material of thisinvention which contains the above mentioned agents may be prepared andused in various forms applicable to dental practice. The dentalrestorative material according to this embodiment may further includeother ions, e.g., antibacterial ions Zn²⁺, Ag⁺, etc. or other additionalingredients depending on the type and form of a particular dentalrestorative material. It is preferable that the pH of dental restorativematerial according to this embodiment be between 2-10, more preferably5-9 and even more preferably 5-7. It is preferable that the pH of thedental restorative material containing the CPP-ACP complex or ACFPcomplex be in the range of about 2 to 10, more preferably in the rangeof about 5 to 9 and even more preferably in the range of about 5 to 7.

According to a further aspect of the invention there is provided avarnish including a compound that is capable of increasing ormaintaining the pH of a solution and a mineralizing agent. Preferably,the mineralizing agent is any stabilized ACP and/or ACFP complex asdescribed herein.

It will be clearly understood that, although this specification refersspecifically to applications in humans, the invention is also useful forveterinary purposes. Thus in all aspects the invention is useful fordomestic animals such as cattle, sheep, horses and poultry; forcompanion animals such as cats and dogs; and for zoo animals.

The invention will now be further described with reference to thefollowing non-limiting examples.

One example of a mineralizing composition or agent comprises thefollowing (in decreasing order of proportion):

-   -   water    -   glycerol    -   CPP-ACP    -   D-sorbitol    -   silicon dioxide    -   sodium carboxymethylcellulose (CMC-Na)    -   propylene glycol    -   titanium dioxide    -   xylitol    -   phosphoric acid    -   guar gum    -   zinc oxide    -   sodium saccharin    -   ethyl p-hydroxybenzoate    -   magnesium oxide    -   butyl p-hydroxybenzoate    -   propyl p-hydroxybenzoate

Such a composition is available from GC corporation under the name ToothMousse™. This is suitable for use after a compound capable of increasingor maintaining the pH of a solution and is in the form of a paste orcrème to facilitate its retention on teeth for a suitable period.Alternatively, this mineralizing composition may contain a compoundcapable of increasing or maintaining the pH of a solution.

Example 1 Preparation of CPP-ACFP and CPP-ACP Solutions

Stock solutions of 3.25M CaCl₂, 1.25 M Na₂HPO₄, 1M NaOH and 1M NaF wereadded in approximately thirty aliquots to a 10-15% w/v tryptic digest ofcasein until a final concentration of approximately 78 mM Ca²⁺, 48 mMphosphate and 12 mM fluoride concentrations were obtained. The solutionswere added slowly (that is, less than approximately 1% volume additionper minute). An aliquot of the phosphate solution was added first,followed by an aliquot of the calcium solution. The pH was maintained at7.0, 6.5, 6.0, 5.5 and 5.0 using the NaOH with thorough mixing. Thesodium hydroxide solution was added automatically by a pH stat with theaddition of the hydroxide ions usually being after each addition of thecalcium ions. After addition of the calcium ions, phosphate ions,hydroxide ions and fluoride ions the solution was filtered through a 0.1micron filter to concentrate 1-2 fold. The retentate may be washed withwater to remove salts and inactive (and bitter tasting) peptides ifdesired. CPP-ACP solutions were prepared as above without the additionof fluoride.

Example 2 Determining Loosely and Tightly Bound Calcium and Phosphate

At the completion of the titration and filtration for each pH in Example1, a sample of each retentate was taken and less than 10% collected as afiltrate using a 3000 molecular weight cut-off Centriprep 3ultrafiltration membrane. The Centripreps containing the samples werecentrifuged at 1,000 g for 15 min in a Beckman J2-21 centrifuge using aJA 10.5 rotor. The original sample before Centriprep centrifugation anda sample of the filtrate after Centriprep centrifugation were taken foranalysis of calcium, phosphate and fluoride concentrations. The analysisof the original sample gave total calcium, phosphate and fluoride ionconcentrations and the analysis of the filtrate gave loosely boundcalcium, phosphate and fluoride concentrations. The difference betweenthe total and loosely bound concentrations is the tightly boundconcentration of Ca, Pi and F by the CPP.

Example 3 Preparation of CPP-ACFP and CPP-ACP Solutions

Recaldent® (CPP-ACP) was purchased from Recaldent Pty Ltd, Victoria,Australia. The product (#841117) contained 14.3% calcium, 22.3%phosphate and 47% casein phosphopeptide on a weight basis. The productwas dissolved at 0.5% and adjusted to pH 5.5 by the addition of HCl.Calcium and phosphate ions were then added by titrating 3.25 M CaCl₂ and2M NaH₂PO₄ while keeping the pH at 5.5 with the addition of 2.5 M NaOH.The titration of calcium and phosphate ions was continued until thesolution became translucent. The concentration of calcium and phosphateadded was recorded. The solution may also be formed by titrating calciumand phosphate ions into a 0.5% CPP-ACP solution and letting the pH fallto 5.5 by the addition of further calcium phosphate.

TABLE 2 Calcium and phosphate levels of normal and superloaded CPP-ACPCalcium Phosphate mmol/L mol/mol CPP mmol/L mol/mol CPP Normal 0.5% 17.822.8 11.6 14.8 w/v CPP-ACP Superloaded 37.8 48.3 23.6 30.2 0.5 w/vCPP-ACP (sCPP-ACP)

These results demonstrate that CPP-ACP can be superloaded with calciumand phosphate ions to produce thermodynamically stable complexes in ametastable solution.

Example 4 Preparation of a Formulation of CPP-ACP and Calcium Phosphate

In another example Recaldent® (CPP-ACP) powder was dry blended withCaHPO4 powder in the ratio CPP-ACP:CaHPO₄ equals 1:10 on a weight basis.This powder was then added to sugar-free gum and toothpaste formulationsat 1-5% w/w.

Example 5

A topical crème may be produced in accordance with the present inventionhaving the following ingredients:

-   -   Water    -   glycerol    -   Stabilized ACP and/or ACFP    -   A compound capable of increasing or maintaining the pH of a        solution    -   D-sorbitol    -   sodium carboxymethylcellulose (CMC-Na)    -   propylene glycol    -   silicon dioxide    -   titanium dioxide    -   xylitol    -   phosphoric acid    -   sodium fluoride    -   flavouring    -   sodium saccharin    -   ethyl p-hydroxybenzoate    -   propyl p-hydroxybenzoate    -   butyl p-hydroxybenzoate

Example 6

A mouthrinse formulation be produced in accordance with the presentinvention having the following composition:

-   -   Water    -   Alcohol    -   Poloxamer 407    -   Sodium Lauryl Sulphate    -   Stabilized ACP and/or ACFP    -   A compound capable of increasing or maintaining the pH of a        solution    -   Sodium Fluoride    -   Flavours    -   Sodium Saccharin    -   Ethyl p-hydroxybenzoate    -   Propyl p-hydroxybenzoate    -   Butyl p-hydroxybenzoate

Example 7

A sugar-free chewing gum formulation be produced in accordance with thepresent invention having the following composition:

-   -   Crystalline sorbitol/mannitol/xylitol    -   Gum base    -   Calcium carbonate    -   Glycerine    -   Stabilized ACP and/or ACFP    -   A compound capable of increasing or maintaining the pH of a        solution    -   Sodium Fluoride    -   Flavour oil    -   Water

Example 8

The effectiveness of the invention could be demonstrated by theexperiments described below. Seven premolar teeth with FLE (ThylstrupFejerskov Index, TF=3) could be selected from teeth extracted fororthodontic reasons from healthy patients aged 10-28 years from theRoyal Dental Hospital of Melbourne, Australia. Informed patient consentshould be obtained for the extracted teeth and the study protocolapproved by the Human Research Ethics Committee of The University ofMelbourne. All specimens should then be debrided of adherent soft tissueand stored in 18% w/v formalin acetate solution at room temperature.

The teeth are then cleaned with a rotating rubber cup and pumice andrinsed in double de-ionized water (DDW) (Fejerskov et al., 1988). Theanatomical crowns are sectioned from the roots using a water-cooleddiamond blade. Each crown is sectioned to provide a pair of enamelblocks each containing a FLE. A 4×4 mm² window should be created overeach lesion by placing a rectangular piece of Parafilm® (AmericanNational Can, Chicago, Ill., USA.) over the lesion and covering thesurrounding enamel with nail varnish (Revion™, New York, USA). Theparafilm would then be carefully removed to reveal the enamel lesionwindow which was divided into halves as control and test windows. Thecontrol window was covered with nail varnish. The two lesions of eachspecimen should be randomly assigned to one of two remineralizationgroups; Group I—treatment with 5% w/v CPP-ACFP or CPP-ACP and GroupII—treatment with 5% w/v CPP-ACFP or CPP-ACP immediately followingpre-conditioning with a compound that is capable of increasing ormaintaining the pH of a solution. Group II could alternatively besimultaneous addition of CPP-ACFP or CPP-ACP with a compound that iscapable of increasing or maintaining the pH of a solution.Alternatively, Group II could be addition of CPP-ACP or CPP-ACFPfollowed by a compound that is capable of increasing or maintaining thepH of a solution.

CPP-ACFP is obtained from Recaldent Pty Ltd (Melbourne, Australia) andcontains 47.6% w/w CPP, 15.7% w/w Ca²+, 22.9% w/w PO₄ and 1.2% w/w F−.The CPP-ACFP is dissolved in distilled and deionized water at 5% w/v andadjusted to pH 7.0 with HCl. For the first group, each specimen shouldbe placed in 2 ml of 5% w/v, CPP-ACFP in a 5 ml plastic vial at 37° C.The CPP-ACFP solution should be changed daily for 10 days. For thesecond group, each specimen should be placed in solution containing acompound that is capable of increasing or maintaining the pH of asolution for 20 mins, rinsed and then placed in 2 ml of 5% w/v CPP-ACFPin a 5 ml plastic vial at 37° C. The CPP-ACFP solution may be changeddaily for 10 days.

A Chroma Meter (Minolta ChromaMeter CR241, Minolta, Japan) is used torecord surface reflectance. Surface reflectance measurement isestablished in L*a*b* color space by the Commission de L'Eclairage in1978, and measurements relate to human colour perception in three colourdimensions (Commision Internationale de L'Eclaige, 1978). The L* valuesrepresent colour gradients from white to black, a* values representcolour gradients from green to red, and b* values represent colourgradients from blue to yellow (Commision Internationale de L'Eclaige,1978). Only L* value measurements can be used with whiter colours havinga higher reading, and darker colours a lower reading. To ensure areproducible position of specimens in the Chroma Meter, a wax mold foreach sample can be prepared and stored. All samples were air-dried witha dental triplex syringe for 60 s before each measurement. Individualspecimens can be repositioned ten times both before and after treatment,and colour reflectance L* values were recorded.

Each specimen is removed from the mineralizing solution and rinsed inDDW for 60 s and blotted dry with blotting paper. The nail varnish onthe control window is removed gently with acetone. The control and testwindows are then separated by cutting through the midline between thewindows. The two half-slabs should then placed with the lesion windowsparallel and embedded in cold curing methacrylate resin (Paladur, HerausKulzer, Germany). The two paired enamel half-slabs are sectioned, andsubjected to microradiography and microdensitometric image analysis todetermine mineral content exactly as described by Shen et al. (2001).

An area free of defects close to the midline of each microradiographicimage of each lesion (control and test) can be chosen and scanned sixtimes (Shen et al., 2001). Each scan comprised 200 readings, taken fromthe enamel surface to the mid-enamel region to include the totalfluorotic lesion. The test (CPP-ACFP-treated) lesion should be scannedto exactly the same depth as the control (untreated) lesion. The grayvalues obtained from each scan are converted to the equivalent thicknessof aluminium (tA) using the image of the aluminium stepwedge includedwith each section (Shen et al., 2001). Using the formula of Angmar etal. (1963), the percentage volume of mineral was obtained for eachreading as follows: V=(52.77(tA)−4.54)/tS. Where: V=volume of mineral asa percentage; tA=the relative thickness of aluminium obtained from thegray value scanned; and tS=section thickness (80 μm).

From the densitometric profile of [(vol % min versus lesion depth (mm)]for each lesion DZ values were calculated using trapezoidal integration(Reynolds, 1997). The difference between the area under the profile ofthe untreated fluorotic enamel in the control window with adjacentnormal enamel is designated DZf, and the difference between the areaunder the CPP-ACFP-treated fluorotic enamel in the test window andadjacent normal enamel is designated DZr. Percentage mineralization (%M) of the fluorotic lesion was therefore (1−DZr/(DZf)×100 (Reynolds,1997).

Following the microradiography the sections containing both control andmineralized FLE can be subjected to Energy Dispersive X-ray Analysis(EDAX) as described previously (Reynolds, 1997). Mean L* values can becompared using a one way classification analysis of variance (ANOVA)with a Scheffe multiple comparison. The mean % M values can also becompared using a one-way ANOVA. Overall mean L* and % M values areanalysed using a paired data Student's t-test.

Example 9 The Effect of Hypochlorite on Enamel SubsurfaceRemineralization by CPP-ACP

Human tooth enamel demineralized subsurface lesions were prepared usingthe method of Reynolds, (1997). J Dent Res 76:1587-1595. The enamelsurface was removed by polishing to remove all adsorbed protein. Thisremoved around 50-100 μm of enamel to expose an uncoated, polishedenamel surface for the study. Furthermore, the acid buffer used toproduce the subsurface lesions did not contain protein such that noprotein would have entered these subsurface lesions. A 1% w/v CPP-ACP pH5.0 solution was prepared using Recaldent® [Kraft Foods], a commercialsource of CPP-ACP that is prepared as per Example 1 at a pH of 5.0. Allblocks were remineralized with the 1% CPP-ACP pH 5 solution for 7 daysat 37° C. However, half were exposed to a 1% v/v sodium hypochloritesolution for 1 min at the start of the CPP-ACP remineralization period.In other words, the sodium hypochlorite and CPP-ACP were simultaneouslyexposed to the subsurface lesion. After remineralization the enamelblocks were embedded, sectioned and subjected to transversemicroradiography and densitometric image analysis as previouslydescribed by Reynolds (1997) to determine percent mineral content gain(% Remineralization) shown in Table 3. A one way analysis of variancewith differences in means determined using a Tukey HSD post hoccomparison showed that the treatment with hypochlorite significantlyincreased the level of remineralization by the CPP-ACP solution by 31%.

Without being bound by any theory or mode of action it is believed thatthe mechanism of enhanced CPP-ACP remineralization by the hypochloriteis related to the diffusion of the hypochlorite into the enamelsubsurface lesions. Once in the lesion fluid it slowly decomposed,thereby releasing chlorine, oxygen, sodium and hydroxide ions.4NaClO+2H₂O→4Na⁺+4OH⁻+2Cl₂+O₂

This slow breakdown of the hypochlorite in the enamel subsurface lesionproduced base (hydroxide ions) which increased the degree of saturationof hydroxyapatite in the intra-lesion fluid as a result of theadditional hydroxide ions. It is believed that the formation ofhydroxyapatite is inhibited by protons which may be released as ACPcoverts to hydroxyapatite or which are present in lower pH environmentssuch as during caries formation. In these ways, the additional hydroxideions, or pH maintained at or above neutral, facilitates the formation ofhydroxyapatite (mineralization) by the CPP-ACP in the lesion. Hencehypochlorite is a non-limiting example of an intra-lesion base producingcompound that will drive remineralization by CPP-ACP or CPP-ACFP.

TABLE 3 Remineralization of Enamel Subsurface lesions by 1% w/v CPP-ACPwith and without 1% v/v Hypochlorite treatment Lesion Treatment DepthΔZd ΔZr ΔZd − ΔZr % Remin. 1% CPP- 109.16 ± 3316.59 ± 2153.24 ± 1163.35± 34.82 ± ACP 11.89 601.49 417.85^(c) 352.97 6.88 1% CPP- 106.65 ±2986.46 ± 1629.30 ± 1357.16 ± 45.48 ± ACP and 16.80 725.39 446.94 365.435.49 1% Hypo- chlorite p-value p < 0.001

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or FIGURES ortables. All of these different combinations constitute variousalternative aspects of the invention.

REFERENCES

-   Fejerskov O, Baelum V, Manji F, Moller I (1988). Dental Fluorosis—A    handbook for health workers Copenhagen: Munksgard.-   Reynolds E C (1998). Anticariogenic complexes of amorphous calcium    phosphate stabilized by casein phosphopeptides: a review. Spec Care    Dentist 18:8-16.-   Reynolds E C, Cai F, Shen P, Walker G D (2003). Retention in plaque    and remineralization of enamel lesions by various forms of calcium    in a mouthrinse or sugar-free chewing gum. J Dent Res 82:206-11.-   Shen P, Cai F, Nowicki A, Vincent J, Reynolds E C (2001).    Remineralization of enamel subsurface lesions by sugar-free chewing    gum containing casein phosphopeptide-amorphous calcium phosphate. J    Dent Res 80:2066-70.

The invention claimed is:
 1. A method of reducing white spot lesions ontooth enamel or subsurface in a patient in need thereof, comprising (i)contacting a white spot lesion with a first composition comprising oneor more mineralizing agents selected from stabilized amorphous calciumphosphate (ACP) and stabilized amorphous calcium fluoride phosphate(ACFP), and (ii) subsequently to (i) and within 1 to 60 minutes of (i),contacting the white spot lesion with a second composition comprising abase, thereby reducing the white spot lesion.
 2. The method of claim 1,wherein the stabilized ACP and/or stabilized ACFP are contacted with thewhite spot lesion for a period of time selected from about 1 minute to 2hours, about 5 minutes to 60 minutes, and about 10 minutes.
 3. Themethod of claim 1, wherein the second composition is contacted with thewhite spot lesion for a period of time selected from about 1 to 60minutes, about 1 to 30 minutes, and about 20 minutes.
 4. The method ofclaim 1, wherein the stabilized ACP and/or stabilized ACFP is repeatedlyapplied to the white spot lesion.
 5. The method of claim 4, wherein thestabilized ACP and/or stabilized ACFP is repeatedly applied to the whitespot lesion over a period of 1 day to several months.
 6. A methodaccording to claim 1, wherein the stabilized ACP and/or stabilized ACFPis in a form selected from a toothpaste, toothpowder, liquid dentifrice,mouthwash, mouthrinse, mouth spray, varnish, dental cement, troche,chewing gum, lozenge, dental paste, gingival massage cream, gargletablet, dairy product, and other foodstuffs.
 7. A method according toclaim 1, wherein the second composition is capable of maintaining the pHof a solution between 7 to
 9. 8. A method according to claim 1, whereinthe second composition is capable of maintaining the pH of a solution atabout 7.5.
 9. A method according to claim 1, wherein the secondcomposition is provided in an amount effective to raise the pH ofintra-lesion fluid of a dental lesion from 6 to 7.5.
 10. A methodaccording to claim 1, wherein the base is selected from sodiumbicarbonate, sodium hypochlorite, and urea.
 11. A method according toclaim 7, wherein the second composition is in a form selected from amouthrinse comprising sodium bicarbonate and a mouthwash comprisingsodium bicarbonate.
 12. A method according to claim 1, wherein thecontacting of step (ii) is effected 1 to 30 minutes after the contactingof step (i).
 13. A method according to claim 1, wherein the contactingof step (ii) is effected 1 to 5 minutes after the contacting of step(i).
 14. A method according to claim 1, wherein the stabilized ACP iscasein phosphopeptide stabilized amorphous calcium phosphate (CP-ACP).15. A method according to claim 1, wherein the stabilized ACFP is caseinphosphopeptide stabilized amorphous calcium fluoride phosphate(CP-ACFP).
 16. A method according to claim 1, wherein the stabilized ACPis casein phosphopeptide stabilized amorphous calcium phosphate (CP-ACP)and the stabilized ACFP is casein phosphopeptide stabilized amorphouscalcium fluoride phosphate (CP-ACFP).
 17. A method according to claim 1,wherein the base is sodium hydroxide.